The replication of the closed-circular, double-stranded plasmid DNA, pBR322, has been reconstituted with purified proteins. The reconstituted system contains: RNA polymerase holoenzyme, ribonuclease H, DNA polymerase I, DNA polymerase III holoenzyme, the single-stranded DNA binding protein, the dnaB, dnaC and dnaG (primase) proteins, proteins i, n, n' (replication factor Y), and n", DNA gyrase and topoisomerase I. Replication proceeds faithfully, according to well-defined criteria based on previous studies in vivo and in vitro on the replication of Co1E1 and pBR322 DNAs, yielding form I DNA product. Major recent findings are that i) DNA gyrase provides the driving force for advance of the replication fork (the known DNA helicases are not required), ii) topoisomerase I imparts specificity to the replication reaction (i.e., restricts initiation to DNA templates containing a pBR322-type origin of replication) and iii) is also required to effect segregation of the daughter DNA molecules. The existence of a replication system completely reconstituted with purified proteins provides the ability to manipulate the system biochemically and orchestrate many partial reactions and subsystems that allow one to study the roles of individual proteins and how these parts sum-up to make the whole. The studies proposed will detail, for the first time, some of the fundamental mechanisms of DNA replication on superhelical DNAs including i) how slight changes in the linking number (induced by topoisomerase I) of the DNA governs initiation of DNA replication, ii) the enzymatic mechanisms involved in the segregation of the daughter duplexes, iii) the pathway of segregation of the daughter molecules, iv) the individual mechanisms for, and the manner of coupling of the synthesis of the leading- and lagging-strand and v) the machinery responsible for unwinding the parental duplex during DNA replication that is under topological constraint.